SCCmec Genotypes of Methicillin-Resistant Staphylococcus aureus in Nasal Carriage of Multiple Sclerosis Patients in Iran

Background: Asymptomatic nasal colonization of Methicillin-Resistant Staphylococcus aureus is common in Multiple Sclerosis patients. SCCmec types I to III are mainly attributed to HA-MRSA strains whereas SCCmec types IV and V have commonly been reported in CA-MRSA infections. Here, we assessed the frequency of nasal carriage of MRSA in MS patients. This study aimed to evaluate MRSA SCCmec typing in MS nasal carriage. Methods: A cross-sectional descriptive study was conducted from Feb and Jun 2017 in MS Research Center, Tehran University of Medical Sciences (TUMS), Iran. Overall, 620 nasal swabs were collected (325 from MS patients and 295 from control group). Antimicrobial susceptibility test was performed using the disk diffusion and E-test method. Presence of mecA gene was confirmed by PCR assay and multiplex PCR was performed for SCCmec typing of MRSA isolates. Results: The frequency of MRSA among the MS patients and control group was almost equal (9.2% and 10.1%, respectively). SCCmec typing detected only types III, IV and V in both groups and type IV was the most predominant type in MS patients and control group. SCCmec type III was more prevalent in control group than MS patients (40% vs. 20%). Moreover, the frequency of SCCmec type V in MS patients was significantly higher than control group (36.7% vs. 3.3%). Conclusion: Although most MRSA isolates were collected from inpatients, interestingly there is a high frequency of SCCmec types IV and V in MS group. Moreover, MRSA isolates were not resistant to more antibiotics in SCCmec type III than types IV–V.


Introduction
Multiple Sclerosis (MS) is an inflammatory demyelinating autoimmune disease that involves the central nervous system (CNS) and usually affects people between the ages of 20 and 50 yr (1). The etiology of MS is currently unknown. Genetic predisposition, T cell activation and environmental factors such as various infections have been contributed to the initiation and exacerbation of this autoimmune disease (2). Infectious diseases appear to play an important role in activating periph-eral autoreactive T cells. Accordingly, development of MS is frequently associated with acquisition of bacterial and viral infections (3,4). Infections caused by Staphylococcus aureus are of serious public health concern throughout the world. S. aureus is one of the major human pathogens, responsible for a wide range of infections from mild to life-threatening conditions (5). This pathogen is increasingly showing resistance to multiple antimicrobial agents (6). Methicillin-resistant Staphylococcus aureus (MRSA) is an opportunistic pathogen characterized by the development in virulence and antibiotic resistance (7,8). MRSA can colonize multiple body sites of human host, but the anterior nares are frequently being colonized in approximately 30%-50% of individuals, moreover, the host can be predisposed to a wide range of infections by asymptomatic carriage of MRSA (9). Nasal colonization of MRSA can be a risk factor for subsequent infection when the balance of host-pathogen is disrupted (10). Although MRSA has been reported as a main hospital-acquired pathogen (HA-MRSA) worldwide, in the last two decades community-acquired methicillin-resistant S. aureus (CA-MRSA) infections has emerged (11). SCCmec types I to III mainly attributed to HA-MRSA strains are associated with multidrug resistance (MDR). SCCmec types IV and V have commonly been reported in CA-MRSA infections (12,13). MRSA originates from methicillin-susceptible S. aureus by acquisition of staphylococcal cassette chromosome mec (SCCmec) element. The SCCmec consists of two essential loci, the ccr gene complex (ccr) and the mec gene complex (mec) harboring the mecA gene. Penicillin-binding protein 2a, encoded by the mecA gene, can cause resistance to methicillin in staphylococci by reduction in affinity (14,15). Molecular typing methods are essential for continuous surveillance and infection control programs, so it may help to prevent the dissemination of MRSA. Among various techniques used for genotyping of MRSA, SCCmec typing is considered as a simple and cost-effective method that can distinguish between the HA-MRSA and CA-MRSA strains (16,17).
Here, we assessed the frequency of nasal carriage of MRSA in MS patients. The aim of this study was to evaluate MRSA SCCmec typing in MS nasal carriage.

Study population
A cross-sectional descriptive study was conducted from Feb and Jun 2017 in MS Research Center, Tehran University of Medical Sciences (TUMS), Iran. Overall, 325 MS patients and 295 apparently healthy individuals, as a control group, were included in the study. Ethical approval for this study was obtained from TUMS ethics committee (Approval number: IR.TUMS.SPH.REC.1396.3054). Samples were collected from persons without MRSA infection. A self-administered questionnaire was provided to each participant to collect demographic information, medical history and factors that potentially may be related to S. aureus nasal colonization and transmission.

Sample processing and identification of bacterial isolates
Samples were collected from the anterior nasal cavities of each patient and healthy person for the isolation of S. aureus using cotton swab. Clinical isolates were cultured on the blood agar and mannitol salt agar and were identified by conventional biochemical tests including catalase, tube coagulase, mannitol fermentation and DNase enzyme production tests. S. aureus isolates were stored in freezer at −70 °C in tryptic soy broth (TSB) containing 20% glycerol.

DNA extraction and identification of MRSA isolates
Genomic DNA was extracted from MRSA isolates by Genomic DNA Extraction Kit (YTA, Tehran, Iran) according to the manufacturer instruction. Isolates were confirmed as MRSA by cefoxitin disc screening test and PCR test for mecA gene.

Multiplex PCR for SCCmec Typing
The standard SCCmec types strains were provided from Sina Molecular Laboratory, Tehran, Iran. Multiplex-PCR assay was performed for the typing of MRSA isolates. Different SCCmec types determined by 9 pairs of primers including the unique and specific primers for ccr gene complex and mec gene complex in order to determine types of SCCmec elements and a pair of primers for the mecA gene (18). Multiplex PCR-1 was optimized by initial denaturation step (94 °C, 2 min), 35 cycles of denaturation (94 °C, 2 min), annealing (57 °C, 1.5 min), extension (72 °C, 2 min), and a final elongation at 72 °C for 2 min and holding at 4 °C. The PCR conditions of Multiplex PCR-2 were as follows: Initial denaturation (94 °C, 1 min), 30 cycles of denaturation (94 °C, 1 min), annealing (60 °C, 1 min), and extension (72 °C, 2 min) and holding at 4 °C. For Multiplex PCR-2, the reaction mixtures are the same as for Multiplex PCR-1 other than the concentration of MgCl2 and the primer pairs. The products were electrophoresed on 1% agarose.

Statistical analysis
Statistical analysis was performed by using Chisquare and Fisher's exact test (SPSS software, version 24, Chicago, IL, USA). A P-value of <0.05 was considered statistically significant. Due to their similar biological features, we considered SCCmec III to be one group and SCCmec IV and SCCmec V to be another group.

Study population
Overall, 620 nasal swabs were collected (325 from MS patients and 295 from the control group). The patient group comprised of 97 (29.85%) males and 228 (70.15%) females. The control group consisted of 104 (35.25%) males and 191 (64.75%) females. The mean age of MS patients and control group was 34.8 ± 11.2 and 30.5 ± 7.7 yr, respectively. Nasal carriage rate of S. aureus among MS patients was 36.3% (n= 118) while in control group it was 32.5% (n=96). There was no statistically significant association between S. aureus colonization and MS disease (P =0.7).

Sample processing and identification of bacterial isolates
The frequency of MRSA among the MS patients and control group accounted for 30 (9.2%) and 30 (10.1%) individuals, respectively. Resistance to cefoxitin was detected in 60 isolates (9.7%). mecA gene amplification was observed in all MRSA isolates. MS patients and control group were not significantly different in the frequency of MRSA colonization (P=0.345). All the MRSA isolates from the study group were susceptible to teicoplanin and vancomycin but relatively resistant to clindamycin and erythromycin. In MS patients group, most isolates were susceptible to rifampicin (90%) and mupirocin (93.4%). In control group, the proportion susceptibility to mupirocin and cotrimoxazole was 90%.

SCCmec Type III vs. Types IV-V in Control Group
However, there was no statistically significant association between SCCmec types and mentioned characteristics. In SCCmec type III group, resistance to some of antibiotics e.g. erythromycin, doxycycline, ciprofloxacin and rifampin were significantly higher than SCCmec types IV-V group.

Discussion
The S. aureus nasal carriage in immunocompromised patients may have serious consequences because nasal colonization can cause an infection when the host-pathogen balance is disturbed. MS patients have impaired T lymphocyte function, then, they are considered as immunocompromised patients. The S. aureus nasal carriage in immunocompromised patients may have serious consequences because nasal colonization can cause an infection when the host-pathogen balance is disturbed (19). The frequency of nasal carriage of S. aureus in MS patients was significantly higher than in healthy persons in other studies (20,21). In present study, nasal carriage rate of S. aureus among MS patients (36.3%) was relatively similar to that of control group (32.5%). The resistance to antibiotics, especially to methicillin, in S. aureus has emerged as a major public health problem. Limited studies were conducted in nasal MRSA carriage in MS patients. The rate of MRSA in Iran was high and ranged from 20.4% to 90% in different areas of the country (22). In current study, the frequency of MRSA was 9.2 % and 10.1% among the MS patients and the control group respectively. Higher rates, 22.2% were reported (20). The frequency of MRSA in MS patients was almost similar to another (9.3%) study (21). The overall rates of SCCmec type were 50%, 30% and 20% for types IV, III and V, respectively. In MS patients, SCCmec type IV (43.3%) was the predominant, followed by type V (36.7%) and type III (20%). In control group, the frequency of types IV, III and V was 56.7%, 40%, 3.3%, respectively. We found that the frequency of type V is significantly higher in MS patients than control group (P=0.001). Although 93.3% (28/30) of MRSA isolates recovered from MS patients were collected from hospital, but surprisingly the molecular typing showed high prevalence of MRSA strains carrying SCCmec types IV and V (91.7%), traditionally attributed to CA-MRSA strains. SCCmec types I to III have been reported to be the most frequent nosocomial MRSA strains in the United States (23,24), Europe (25,26), and Switzerland (27,28). In southern Iran, type III was the most prevalent (74.3), followed by type IV (12.1%), type V (2.6%) and type I (0.6%) (29). In another study in Iran, SCCmec types III (45%) and IVc (24%) were the most prevalent among HA-MRSA isolates (30). In this study, we considered SCCmec III to be one group and SCCmec IV and SCCmec V to be another group (due to their similar biological features) and compared the demographic characteristics and antibiotics susceptibility pattern between two groups, in MS patient and control group, separately. Like other studies from Iran, most isolates were sensitive to vancomycin (100%) and teicoplanin (100%). The highest rate of resistance was observed for clindamycin and erythromycin. In MS patients group, resistance to clindamycin, doxycycline and rifampin was significantly higher in SCCmec type III vs. types IV-V group. In control group, resistance to erythromycin, doxycycline, ciprofloxacin and rifampin group was significantly higher in type III than in types IV-V group. Overall, the antimicrobial resistance patterns in MS patients and control group were relatively similar. In Iran, decreased sensitivity of HA-MRSA to erythromycin, clindamycin, co-trimoxasol, ciprofloxacin, cephalexin, tetracyclin, and gentamicin, predorminatly were reported among isolates harboring type III SCCmec gene (30). In Iran, all HA-MRSA isolates were susceptible to quinupristin-dalfopristin, linezolid, and vancomycin, but most isolates were resistant to penicillin (100%), erythromycin (50%), clindamycin (27%), and gentamicin (18%) (29). Vancomycin, quinupristin-dalfopristin, linezolid and teicoplanin can still be used to treat different MRSA infections in Iran. In MS patients and control groups there is no statistically significant association between SCCmec types groups and demographic characteristics include, hospitalized patients, average age, history of antibiotic usage, nasal and upper respiratory failure.

Conclusion
Although most MRSA isolates were collected from inpatients, interestingly there is a high frequency of SCCmec types IV and V in MS group. Moreover, MRSA isolates were not resistant to more antibiotics in SCCmec type III than types IV-V.

Ethical considerations
Ethical issues (Including plagiarism, informed consent, misconduct, data fabrication and/or falsification, double publication and/or submission, redundancy, etc.) have been completely observed by the authors.